In Situ Analysis of DNA Damage Response and Repair Using Laser Microirradiation

Kim, Jong‐Soo; Heale, Jason T.; Zeng, Weihua; Kong, Xiangduo; Krasieva, Tatiana B.; Ball, Alexander R.; Yokomori, Kyoko

doi:10.1016/s0091-679x(06)82013-3

Cited by 18 publications

(21 citation statements)

References 79 publications

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“…To further investigate the localization of Sp1 to sites of DNA DSBs and determine the kinetics of recruitment, we used laser microirradiation to induce DSBs in defined areas of the nucleus (39). Cells were processed for immunofluorescence at various time points after laser treatment.…”

Section: Resultsmentioning

confidence: 99%

Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism

Beishline

Kelly

Olofsson

et al. 2012

Molecular and Cellular Biology

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cSp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H 2 O 2 . Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant ␥H2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects.T ranscription factor Sp1 regulates the expression of genes involved in cell proliferation, DNA repair, and apoptosis/ senescence (9). DNA binding of Sp1 is mediated through three zinc fingers in the C-terminal region, which recognize GC-rich elements in a large number of promoters that are frequently found in euchromatic CpG islands. Posttranslational modifications throughout Sp1, including phosphorylation, acetylation, O-linked glycosylation, and sumoylation, modulate its interaction with chromatin remodeling factors, DNA, transcription machinery, and other transcription factors to enhance or repress gene expression (13,35,36,40,51,53,56,69,82). Our group and others have shown that transcription factor Sp1, which contains two S/TQ cluster domains (SCDs), characteristic of phosphoinositide 3-kinase-like kinase (PI3KK) substrates, is phosphorylated by the ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation, H 2 O 2 (64), and other DNA-damaging agents (unpublished data), as well as herpesvirus infection (33).Genomic stability is maintained by the cellular response to DNA damage. In response to DNA double-strand breaks (DSBs), ATM is activated (80) and initiates a cascade of DNA damage signals by phosphorylation of hundreds of proteins involved in cell cycle checkpoint activation, DNA repair, and apoptosis, including p53, Chk2, ␥H2Ax, BRCA1, and Nbs1 (48, 57). Mutations in the ATM gene result in the autosomal recessive disorder ataxia telangiectasia (AT), which is characterized by radiation sensitivity, immunodeficiency, neurodegeneration, and cancer predisposition (79). Cells derived from AT patients exhibit increased chromosome breaks, defects in cell cycle checkpoints, and increased sensitivity to ionizing radiation (IR) (66,78). Inactive ATM forms...

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Section: Resultsmentioning

confidence: 99%

Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism

Beishline

Kelly

Olofsson

et al. 2012

Molecular and Cellular Biology

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“…Another consideration is the use of photosensitizers, like halogenated nucleotide analogues (e.g., BrdU) or DNA binding dyes (e.g., Hoechst), to lower the energy required to induce DNA strand breaks. Another consideration is that these agents can cause undesirable effects in chromatin structure that could alter the kinetics of DNA damage response, and may have additional damaging effects during long term imaging with visible light (5, 6, 11, 39). These effects are most often observed in the kinetics of DSB repair followed by live cell imaging, yet they are important to consider when evaluating the application of a laser technique.…”

Section: Uv Wavelengths To Induce Of Dna Damagementioning

confidence: 99%

Micro-irradiation tools to visualize base excision repair and single-strand break repair

Gassman

Wilson

2015

DNA Repair

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Microscopy and micro-irradiation imaging techniques have significantly advanced our knowledge of DNA damage tolerance and the assembly of DNA repair proteins at the sites of damage. While these tools have been extensively applied to the study of nucleotide excision repair and double-strand break repair, their application to the repair of oxidatively-induced base lesions and single-strand breaks is just beginning to yield new insights. This review will focus on examining micro-irradiation techniques reported to create base lesions and single-strand breaks; these lesions are considered to be primarily addressed by proteins involved in the base excision repair (BER) pathway. By examining conditions for generating these DNA lesions and reviewing information on the assembly and dissociation of repair complexes at the induced lesion sites, we hope to promote further investigations into BER and to stimulate further development and enhancement of these techniques for the study of BER.

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“…We used laser micro-irradiation to introduce damage and monitored the recruitment of GFP-tagged PRC2 components to DNA damage sites in real-time. This approach is commonly employed to study the recruitment of DSB repair proteins, 13,15,23,24 although it introduces other types of DNA damage as well. Laser micro-irradiation involves first sensitizing cells with a DNA intercalator, such as Hoechst.…”

Section: Prc2 Proteins Recruit To Laser Microirradiation-induced Dna mentioning

confidence: 99%

Polycomb repressive complex 2 contributes to DNA double-strand break repair

et al. 2013

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Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (eZH2), is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether eZH2 has a role in modulating the DNa damage response. Here, we show that polycomb repressive complex 2 (PrC2) is recruited to sites of DNa damage. this recruitment is independent of histone 2a variant X (H2aX) and the PI-3-related kinases atM and DNa-PKcs. We establish that ParP activity is required for retaining PrC2 at sites of DNa damage. Furthermore, depletion of eZH2 in cells decreases the efficiency of DSB repair and increases sensitivity of cells to gammairradiation. these data unravel a crucial role of PrC2 in determining cancer cellular sensitivity following DNa damage and suggest that therapeutic targeting of eZH2 activity might serve as a strategy for improving conventional chemotherapy in a given malignancy.

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In Situ Analysis of DNA Damage Response and Repair Using Laser Microirradiation

Cited by 18 publications

References 79 publications

Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism

Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism

Micro-irradiation tools to visualize base excision repair and single-strand break repair

Polycomb repressive complex 2 contributes to DNA double-strand break repair

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